One of the major challenges in the treatment of cancer is the large variety of oncogenic alterations within the cancer genome affecting many different cellular processes. As such, effective therapy will likely be based on the specific genetic alterations present in each patient's tumor rather than a single generalized therapy regimen. I recently discovered frequent inactivating mutations of genes including STAG2 that encode the multi- protein cohesin complex in several tumors including glioblastoma, urothelial carcinoma, Ewing sarcoma, and acute myeloid leukemia (AML), which define molecular subgroups of these tumors with distinct clinical outcomes. The cohesin complex is responsible for sister chromatid cohesion following DNA replication and helps ensure faithful chromosome segregation during mitosis, but has also been implicated in additional cellular processes such as regulation of chromatin architecture and gene transcription. My studies in glioblastoma demonstrated that STAG2 mutations were a direct cause of chromosomal instability and aneuploidy; however, cohesin gene alterations in urothelial carcinoma and AML have been identified primarily in near-diploid tumors, suggesting alternative mechanisms by which cohesin inactivation drives oncogenesis. Using a newly generated conditional STAG2 knockout mouse and previously generated isogenic sets of STAG2 proficient and deficient cancer cell lines, I have designed a series of experiments to determine the function of STAG2 during mouse development and tumorigenesis and identify therapeutic methods to selectively target cancer cells harboring cohesin gene mutations. In the first aim, I will determine the impact on murine development of inducing STAG2 knockout starting in embryogenesis using both histologic and cellular characterization. In the second aim, I will determine the function of STAG2 in urothelial carcinogenesis and gliomagenesis by inducing specific knockout of STAG2 within the epithelium of the urinary tract and glial cells of the central nervous system. The proposed studies will prove if STAG2 is a bona fide tumor suppressor gene, identify cooperating genetic alterations, and determine whether chromosomal instability is a driving oncogenic mechanism. In the third aim, I will determine possible alternative mechanisms by which STAG2 inactivation contributes to tumor development. I will perform experiments to assess the hypothesis that cohesin localizes to regulatory regions of genes that control cellular differentiation and that STAG2 inactivation disrupts cohesin- mediated regulation of these genes controlling differentiation. In the final aim,I will use isogenic sets of STAG2 proficient and deficient cells to determine the role of cohesin in DNA damage repair and the response to radiation and specific DNA damaging agents, as well as perform a CRISPRi-based negative selection screen in order to identify gene products that are required (i.e. synthetically lethal) in the presence STAG2 inactivation. Together, these experiments will determine the function of STAG2 in mouse development and tumorigenesis and identify therapeutic vulnerabilities in the many cancers harboring cohesin gene alterations.